RESEARCH ARTICLE

Hypersalinity drives physiological and morphological changes inLimia perugiae (Poeciliidae)Pablo F. Weaver1,*, Oscar Tello1, Jonathan Krieger2, Arlen Marmolejo3, Kathleen F. Weaver1, Jerome V. Garcia1

and Alexander Cruz4

ABSTRACTA fundamental question in biology is how an organism’s morphologyand physiology are shaped by its environment. Here, we evaluate theeffects of a hypersaline environment on the morphology andphysiology of a population of livebearing fish in the genus Limia(Poeciliidae).We sampled from two populations ofLimia perugiae (onefreshwater and one hypersaline) in the southwest DominicanRepublic.We evaluated relative abundance of osmoregulatory proteins usingwesternblot analysesandusedageometricmorphometric approach toevaluate fine-scale changes to size and shape. Our data show that gilltissue isolated fromhypersaline fishcontainedapproximately twoandahalf times higher expression of Na+/K+ ATPase proteins.We also showevidence for mitochondrial changes within the gills, with eight timesmore complex I and four times higher expression of ATP synthasewithin the gill tissue from the hypersaline population. The energeticconsequences to Limia living in saline and hypersaline environmentsmay be a driver for phenotypic diversity, reducing the overall body sizeand changing the relative size and shape of the head, as well asimpeding the growth of secondary sex features among the males.

KEY WORDS: Poeciliidae, Hypersalinity, Osmoregulation,Mitochondria, Geometric morphometrics

INTRODUCTIONLivebearing freshwater fishes in the genus Limia (Poeciliidae) arediverse in their morphology, behavior, and ecology. In terms ofoverall species diversity and distribution, the genus Limia is one ofthe dominant freshwater fish groups in the West Indies, withupwards of 17 described endemic species occurring on Hispaniolaand one endemic species on Cuba, Grand Cayman, and Jamaica(Rauchenberger, 1988; Burgess and Franz, 1989; Rodriguez, 1997;Hamilton, 2001). The broad ecological tolerances and adaptabilityto varied environments exhibited by these species are likely key toboth their colonization and diversification in the West Indies.Hispaniola, the center of biodiversity for Limia, provides a

natural laboratory to examine the roles of environmental change in

driving divergent evolution. While second in land area to Cuba,Hispaniola is considered to be more topographically diverse, havingboth the highest point in the Caribbean (Pico Duarte, 3117 m abovesea level) and the lowest point (near Lago Enriquillo, 45 m belowsea level). Limia species exist on all corners of the island, occupyinga diversity of aquatic habitats, from cool mountain streams to warmcoastal lagoons. The largest assemblage of Limia species onHispaniola is distributed mainly in the southwest Cul de Sac andValle de Neiba region near Lago Enriquillo in the DominicanRepublic. Across this region, Limia species exhibit theiradaptability by their presence in both freshwater, as well as salinehabitats. Several populations even occupy hypersalineenvironments in the region (Burgess and Franz, 1989).

Here we investigated the role of salinity in driving physiologicaland morphological change in Limia. We focused on the speciesL. perugiae Evermann and Clark, 1906, which is one of the mostsuccessful and widely distributed species in the Limia genus.Populations of L. perugiae occupy freshwater ecosystems, as well assaline [∼35 parts per thousand (ppt)] and hypersaline (40-100 ppt)lakes and coastal lagoons. In the southwestern region of theDominican Republic, three hypersaline areas are occupied byL. perugiae: Lago Enriquillo, Laguna de Oviedo, and the LasSalinas/Las Calderas area ∼100 km SW of Santo Domingo. Allthree habitats fluctuate in their salinity (ranging from 40-100 ppm)but are by nature consistently hypersaline (Buck et al., 2005). In allthree hypersaline localities, L. perugiae individuals are known to besmaller and less ornate than their neighboring freshwaterpopulations (P.F.W., unpublished). The goals for this work wereto evaluate the role of hypersalinity in driving phenotypic diversity.Specifically, we were interested in identifying the changes tomitochondrial energy production and trans-membrane ion transportmechanisms in the gills and how these may relate to largermorphological changes observed in the field.

Studies show that fish growth and development are dictated bytradeoffs in energy demand and are directly fueled by energysurplus (Ricker, 1975; Chen et al., 1992; Lester et al., 2004).Energy obtained from food is primarily divided into the processesof metabolism, excretion, ion/osmoregulation, and primaryreproductive features. The difference between the energy inputand the primary allocations of that energy is considered surplusenergy, which can then be applied to growth and the development ofsecondary reproductive features. Extensive research has attemptedto quantify and predict the growth of fish, based on changes inenergy allocation caused by various changes in the environment,such as temperature, water chemistry, and feed (e.g. VonBertalanffy, 1957; Lester et al., 2004). The amount of energy thatmay be allocated for growth can be depicted by the followingtheoretical equation, adapted from Lester et al. (2004):

Eg ¼ Ef �ðEm þ Eo=e þ EPrÞ�ESr: ð1ÞReceived 26 January 2016; Accepted 22 June 2016

1Department of Biology, University of La Verne, 1950 3rd St., La Verne, CA 91750,USA. 2Herbarium, Library, Art & Archives Directorate, Royal Botanic Gardens, Kew,Richmond, Surrey TW9 3AE, UK. 3Instituto de Investigaciones Botánicas yZoológicas Prof. Rafael M. Moscoso, Universidad Autónoma de Santo Domingo,Santo Domingo, Dominican Republic. 4Department of Ecology and EvolutionaryBiology, University of Colorado, Boulder, CO 80309-0334, USA.

*Author for correspondence (pweaver@laverne.edu)

P.F.W., 0000-0003-1576-1366

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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In this equation, energy for growth (Eg) is dependent on the differencebetween the energy obtained from food (Ef ) and the essential energyrequirements, the minimal energy requirements from metabolism(those needed for the organism to survive; Em), osmoregulation/excretion (Eo/e) and primary reproduction (EPr), and the energyneeded for development of secondary reproductive features (ESr).Under this growth model, any change to the energy demands of

survival decreases the amount of energy that may be allocated forgrowth. Studies have demonstrated that changes in environmentalfactors, such as water quality and temperature can elicit a decrease insize as a result of the allocation of energy towards processesinvolved with survival in that environment (Lema and Nevitt, 2006;Horppila et al., 2011; Muñoz et al., 2012). The mechanism forsomatic growth is so energy-sensitive that, even at optimalconditions, reproductive needs associated with maturation inhibitsfurther growth. Lester et al. (2004) found that during the pre-maturity stage of development, surplus energy is more likely to beallocated towards growth than during the sexually mature adultstage. Reproductive costs (production of spermatozoa or eggs andmate searching) deplete the surplus energy store, leaving less energyfor growth. Thus, any deviation in energy expenditure mayinfluence fish morphology. Drastic changes in environment, suchas colonizing a hypersaline lagoon, have the potential to createsubstantial deficiencies of energy.Osmoregulation in fish is complex and potentially energetically

expensive. Previous studies estimate energetic costs at anywherefrom 1% to 50% of the energy budget, depending on the tonicity ofthe environment (Rao, 1968; Nordlie and Lefler, 1975; Nordlie,1978; Furspan et al., 1984; Nordlie et al., 1991; Toepfer and Barton,1992; Evans, 2008; Ern et al., 2014). The primary modulators ofions in fish gills are the mitochondria-rich cells, which are the site ofthe transmembrane ion transport proteins, specifically sodiumpotassium ATPase (Na+/K+ ATPase) (Evans et al., 2005). Na+/K+

ATPase proteins use energy in the form of adenosine triphosphate(ATP) to move sodium ions against their concentration gradient.Previous work has demonstrated substantial changes to theosmoregulatory mechanisms of fish in hypersalinity, at themolecular level, tissue morphology, and organismal physiology(Stuenkel and Hillyard, 1980; Sardella et al., 2004; Bystriansky

et al., 2007). For example, one of the primary predictable changesthat occurs in most euryhaline teleost fishes is an increase inbranchial Na+/K+ ATPase activity as a way of maintainingosmoregularity in an increasingly saline environment (reviewedby Sakamoto et al., 2001; Marshall, 2002).

Given the energetic demands of osmoregulation and the need forsurplus energy for normal growth and development, we can predictthat hypersaline environments provide a strong selective pressureon their inhabitants. The colonization of hypersaline waters byL. perugiae offers an opportunity to examine morphologicalchanges, as well as physiological responses to an extremeenvironment and to evaluate populations potentially undergoingdivergent natural selection. For fish in hypersalinity, we predictedincreased energy demands would lead to changes to theosmoregulatory mechanisms, and that morphological changeswould reflect both physiological and environmental differences.

RESULTSThe water conditions from the hypersaline and freshwater localities(Figs 1 and 2) differed in all environmental measurements, althoughthey were most different in salinity, with the Las Salinas(hypersaline) water measuring 42.08 ppt compared with 0.33 pptfor the La Raqueta (freshwater) site (Table 1). Sites also differed intheir dissolved oxygen, with 6.09 mg/l at Las Salinas and 8.12 mg/lat La Raqueta.

PhysiologyFor this study, mitochondrial and ion transport proteins wereanalyzed from hypersaline and freshwater fish gills (Fig. 3).Densitometry results showed that fish gills isolated fromhypersaline individuals contained eight times more mitochondrialcomplex I protein, represented by a band at ∼30 kDa, a fourfoldhigher expression ofATP synthase, represented bya band at∼60 kDaand finally a two-and-a-half-fold increase in expression of Na+/K+

ATPase proteins, represented by a band at ∼100 kDa. All threeproteins probed are made up of many subunits, and the antibodiesused recognize specific subunits of the protein. The Complex Iantibody recognizes the anti-NDUFS3 the iron-sulfur subunit that hasa predicted band size of 30 kDa, the ATP synthase antibody

Fig. 1. Map of localities for populations of L. perugiae inthe southwest Dominican Republic. Hypersalinepopulations are indicated by open circles and nearbyfreshwater populations are represented by shaded triangles.The two populations used in this study are indicated by theoval.

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recognizes the alpha subunit (anti-ATP5A), which has a predictedband size of 60 kDa, and Na+/K+ ATPase antibody recognizes theanti-Na+/K+ ATPase α1 subunit that has a predicted band size of100 kDa. Bands that depict the specific subunits, when compared tothe molecular weight markers, have comparable weights to thepredicted band size. Beta actin was used as a loading control andrepresented by a band at 45 kDa. Although the data show thathypersaline fish have increasing concentrations of Complex I, ATPsynthase, and Na+/K+ ATPase proteins, as determined by the densityof the bands, it cannot be determined whether or not this increase inconcentration is due to an increase in protein synthesis, from either anincrease in mitochondrial content or matrix restructuring of the pre-existing mitochondria, or to a decrease in protein degradation.

Geometric morphometricsIn total, we examined 86 individuals from the two sites for thegeometric morphometric analysis. Freshwater fish from La Raquetaaveraged 3.01±0.79 cm (mean±s.d.) in length and 1.02±0.23 g inmass, and fish from Las Salinas averaged 2.30±0.51 cm in lengthand 0.27±0.05 g in mass. A plot of the first two principalcomponent axes from the combined dataset of landmarkcoordinates, including males and females shows that individualscollected in the hypersaline habitat are a distinct ecotype and occupy

a different area in morphospace, with lesser scores along PC2, andespecially along PC1 (Fig. 4). Differences between populations aremore drastic for males than females, but vary in the same way (themeans diverge along the same angle with respect to PC1 and PC2).Additional analyses of the male only dataset allowed us to furtherexamine specific aspects of shape that varied between thehypersaline and freshwater ecotypes. In this case, differencesbetween populations can be best observed by PC1 and PC3, with theindividuals collected in the hypersaline habitat clumping with lesserscores along PC1 and higher scores along PC3 (Fig. 5).

We ran post hoc tests using GLHT, holding the centroid sizeconstant. Results show that the hypersaline males vary significantlyfrom the freshwater males along ES3 (P

PhysiologyAs predicted, we observed an upregulation of the active ion transportprotein Na+/K+ ATPase in the hypersaline population. Na+/K+

ATPase has been shown to be the key transport mechanism for

sodium ions across themembrane and is observed to be upregulated ineuryhaline fishes acclimating to progressively saltier waters (Langdonand Thorpe, 1984; Evans et al., 2005; Seo et al., 2009). The drivingsource of energy for active ion transport, as with all cellular processes,

Fig. 4. Scatter plot of relative warp scores along the first two principal component axes. The hypersaline population from Las Salinas is indicated by openshapes, and the freshwater population from La Raqueta is indicated by closed shapes. Males are indicated by triangles, females by circles. The individuals mostclosely representing the means are circled. Shape differences can be seen between sexes, as well as between habitat type.

Fig. 5. Scatter plot of relativewarp scores along the twomost significant principal component axes (PC1 and PC3) from the MANOVA on the male onlydataset. The hypersaline population is indicated by circles and the freshwater population by triangles. Deformation grids showing the specific changes in shapealong each axis are shown. Individuals from both populations that are closest to the mean are circled.

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is the mitochondria. Energy demands caused by environmentalchange, such as temperature and salinity, could in concept triggermitochondrial remodeling, an increase in mitochondria per cell, or anincrease in the number of chloride cells in order to accommodateincreased bioenergetics needs. The main source of ATP production inthe mitochondria is the electron transport system. We focused oncomplex I because it can be a large contributor to total flux through theelectron transport system. Therefore, Complex I has a large influenceon the chemo-osmotic potential necessary to drive the ATP synthasecomplex. Here we show an eightfold increase in Complex I in the gilltissues of hypersaline fish. We also show a fourfold increase in ATPsynthase, used to catalyze the production of ATP from ADP. Bothprocesses can be seen as critical mechanisms involved in adjusting tothe increased demands of osmoregulation. As Na+/K+ ATPaseproteins are upregulated and are involved directly in the activetransport of sodium across the membranes, Complex I and ATPsynthase provide the additional fuel necessary to complete the process.In relation to our original growth equation (Eqn 1), our physiologyresults indicate increased energy allocation towards metabolism (Em)and osmoregulation (Eo/e), with subsequent predicted effects onoverall growth, reproduction, and development of secondaryreproductive features.

MorphologyA fundamental question in evolutionary biology is how anorganism’s phenotype changes in response to its environment.

A growing area of research evaluates populations undergoingdivergent natural selection to identify the drivers of phenotypicdiversity, which are key to the process of ecological speciation(Schluter, 2001; Funk et al., 2002, 2006; Rundle and Nosil, 2005).Studies of divergent natural selection among fishes have mainlyfocused on biotic interactions (e.g. Endler, 1986; Langerhans andDeWitt, 2004; Langerhans, 2009) and how predator regimesinfluence features such as coloration, body shape, performance,and life history characteristics. Research has also begun to evaluatethe roles of abiotic selective pressures, such as temperature (Lemaand Nevitt, 2006; Muñoz et al., 2012; Nilsson et al., 2009), toxins(Riesch et al., 2010; Tobler et al., 2011), and salinity (Boeuf andPayan, 2001; Horppila et al., 2011) in driving population leveldifferences in the morphology and physiology of fishes. Extremeenvironments should amplify the effects of divergent naturalselection, as seen with livebearing fishes in the toxic sulfur poolsof Mexico (Tobler et al., 2011).

Our results indicate that the divergent natural selective pressuresof the two environments, may be driving phenotypic diversificationin L. perugiae, including body size, mass, and shape change. Inaddition to differences in salinity, it is evident that the more salinehabitat is alsowarmer (31°C vs 27°C). If one assumes Q10 values of2-3 for biological rate functions, this could result in a 30%-60%greater metabolic demand irrespective of salinity (Lema and Nevitt,2006). The combination of temperature and salinity is likely apowerful selective force in this environment.

Fig. 6. Morphometric differences between the freshwater andhypersaline ecotypes. Values are from the outputs box plots showthe differences along the seven principle component axes. Differencein centroid size (CS) is also shown.

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Due to the sensitivity of a fish’s overall energy balance, anyadaptations to osmoregulatory mechanisms or other energeticdemands may be diverting energy away from growth anddevelopment. Although an increase in mitochondrial respiration inthe gills of hypersaline fish may accommodate this extra demand,this only underscores the shifting of energy resources towards thegill area (to feed active transport) and away from other bodily needs.Overall, the morphological feature to change the most was bodysize, with fish from the hypersaline population measuring roughlythree quarters the size and mass of the freshwater counterparts.Another observable difference between populations was the relativesize of the head, with the hypersaline fish exhibiting proportionallylarger heads than the freshwater fish (Figs 4, 5 and 7). Similarpatterns have been seen in fishes inhabiting toxic sulfidicenvironments (Tobler et al., 2011). As in the sulfidic environmentstudy system, the larger head and gill space may be an adaptation toincreased respiratory demands of an extreme environment.Morphological change can be seen most dramatically with males,

who differ most in overall body size, as well as some otherinteresting shape features. Freshwater males develop distinctsecondary sex features, including a broader body and elongatedfins, used in courtship display behavior (Farr, 1984; Applebaum andCruz, 2000; Munger et al., 2004). While we did not include finshape analysis in this study, differences in overall body shape areevident, with hypersaline males being narrower bodied and morejuvenile in appearance. An interesting follow up to this study wouldbe to study population dynamics in the wild as well as to conductbehavioral experiments to evaluate differences in both malebreeding behavior and female choice. Behavioral experimentswould allow us to evaluate whether sexual selection is a factor in thehypersaline environments, and more importantly, whetherreproductive isolating mechanisms, a precondition for ecologicalspeciation (Schluter, 2001; Langerhans, 2009), may be developingin these populations.An important question, and one that will need to be answered in

order to evaluate the true significance of our results, is whether thechanges we have seen are truly adaptations in an evolutionary sense,

or whether these changes are due to phenotypic plasticity, as seen inthe gills of salmonids and eels, and the morphology of three spinesticklebacks as they transition between salinity gradients(Mazzarella et al., 2015). It cannot be ruled out that changes tothe ion transport proteins and mitochondrial respiration are highlyinducible in fish and that L. perugiae can be cross acclimated to anopposite salinity regime (P.F.W., unpublished). This potentialunderscores the need for further laboratory studies on thedifferences between a fish’s acclimatization response and longterm adaptation. Studies on tilapia have shown phenotypic plasticresponses in gill morphology under hypoxia, as well as geneticassimilation of those changes over generations (Chapman et al.,2000). Heritable change is also supported through our own evidencefor lab-reared generations of hypersaline populations maintainingthe dwarfed appearance of their wild-caught ancestors.

In conclusion, the Limia of the West Indies are a showcase forevolution and adaptation to novel environments. While poeciliidscontinue to provide fertile ground for studies of ecologicalspeciation, little work has evaluated the physiological/biochemicalchanges that underlie the mechanisms for adaptation. L. perugiaeoffers a new system to evaluate some of the potential inner workingsof adaptations under divergent natural selection.

METHODSFish samplingAll experiments were conducted under guidelines and protocols approvedby the IACUC committee at the University of La Verne, and fish weresampled and transported with appropriate permits from wildlife agencies inthe Dominican Republic. Fish used in this experiment were collected in thewild from the Las Salinas area of the Dominican Republic in December2013 (Figs 1 and 2). Adult fish of both sexes were randomly sampled from

Table 2. Results of the post hoc-adjusted mean Tukey GLHT testsevaluating differences between freshwater and hypersalinepopulations for the seven principal components of shape

Source Estimate s.e.m. T-value P-value

PopulationES1 −0.008527 0.010756 −0.79 0.432PopulationES2 −0.000671 0.008414 −0.08 0.937PopulationES3 0.013679 0.005665 2.42 0.019*PopulationES4 0.002589 0.005043 0.51 0.61PopulationES5 0.010752 0.004088 2.63 0.011*PopulationES6 −0.006283 0.003852 −1.63 0.109PopulationES7 −0.007900 0.003363 −2.35 0.023*

*P-values significant at

the wild in each of the following areas: ‘hypersaline’ individuals werecollected from the Las Calderas lagoon (18.2125 N, 70.5397 W) and‘freshwater’ individuals were collected near the neighboring town of LaRaqueta (18.2311 N, 70.3601 W).

Environmental variables of water temperature, salinity, pH, and dissolvedoxygen were measured using a YSI Model 556 multi probe (YSI Inc.,Yellow Springs, OH USA). All fish were caught using seine nets and weretransferred to 37-liter holding containers of their native waters. Live fishwere then transported to the University of La Verne for western blotanalysis.

Western blottingPrevious research has demonstrated that during the process of acclimating tohigher salinity, fish significantly increase both the number and activity ofNa+/K+ ATPase proteins, as well as sodium/potassium chloride co-transporters (Scott et al., 2004; Tseng and Hwang, 2008; Tse and Wong,2011). As the upregulation of the two proteins are coupled, and ultimatelydependent on the pumping of Na+ outside the cell by Na+/K+ ATPase, wefocused on the Na+/K+ ATPase protein (alpha 1 subunit) and the changesinduced in the biosynthesis of ATP via aerobic metabolism, specificallyComplex I (NDUFS3 subunit) and ATP synthase (alpha subunit of ComplexV, one of the 18 subunits encoded by the nuclear and mitochondrial genes).Three fish were randomly sampled from each locality (hypersaline andfresh) and were sacrificed with anMS-222 (3-aminobenzoic acid ethyl ester)(Sigma, St. Louis, MOUSA) overdose, after De Tolla et al. (1995) and Reedand Jennings (2010). The gills were excised and homogenized in SEIBuffer, containing 0.3 M sucrose, 0.02 M EDTA, 0.1 M imidazole, and0.1% CHAPS. Proteins were isolated via perchloric acid precipitation, andconcentration was determined using a NanoDrop Lite Spectrophotometer(Thermo Scientific, Waltham, MA USA). 150 mg of protein was placed in1× Pierce Lane Marker Reducing Sample Buffer (#3900; ThermoFisherScientific, Inc., Grand Island, NY USA), loaded with saltwater fish on oneside of the gel and freshwater fish on the other, and resolved in 10% SDSPAGE. The protein was then transferred to a polyvinylidene fluoride(PVDF) membrane, blocked with casein (#161-0782; Bio-Rad, Hercules,CA USA), and probed with Complex I, ATP synthase, and Na+/K+ ATPaseantibodies for two hours at room temperature. Mitochondrial antibodieswere obtained from Mitosciences (Eugene, OR USA) against Complex I(anti-NDUFS3; 1:2000; ms110-ab110246 mouse monoclonal antibody)and ATP synthase [anti-ATP5A (the alpha subunit); 1:1000; ms502-ab110273 mouse monoclonal antibody). Anti-Na+/K+ ATPase α1 subunitantibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,CA USA) (1:1000; sc-16043 goat polyclonal antibody). Manufacturers’protocols lists zebrafish proteins as cross-reactive with all antibodies. Toensure equal loading, the gel was probed with a monoclonal mouse anti-betaactin antibody (1:1000; ab170325; Abcam, Cambridge, MA USA). Theanti-actin antibody was incubated for two hours at room temperature.Membranes were washed and blocked again with casein, then probed with asecondary goat anti-mouse (sc2005) or donkey anti-goat antibody (sc2020;both at 1:2500) secondary antibodies from Santa Cruz Biotechnology, Inc.,for one hour at room temperature. Chemiluminescence was captured with

Hyblot CLAutoradiography Film (e3012) obtained fromDenville ScientificInc. (Holliston, MA USA) and band intensity compared between samplesusing Image Studio Lite Software package (version 3.1).

Geometric morphometricsMass and standard length of fish from nose to base of the caudal fin wererecorded for all individuals. We used landmark-based geometricmorphometrics to quantify phenotypic differences between individualsfrom the freshwater and hypersaline populations. Previously sacrificed fish,whose gills had been excised and used for protein analysis, wereimmediately prepped for photography. Additional individuals weresacrificed in MS222, so that overall we photographed 29 males and 16females from the freshwater La Raqueta population and 29 males and 12females from the saltwater Las Salinas population. Our sampling effort herewas as high as possible to increase statistical power, with considerationgiven to not oversampling from the population. Only mature adults wereincluded in the analysis. High resolution images were taken with a Nikon(Tokyo, Japan) 7000 camera body and a 105 mm Nikkor macro lens,following the protocol of Schutz and Krieger (http://www.morpho-tools.net/softwareguide/GM%20guide%20v4%20OLs.pdf ). Sixteen landmarks(Fig. 8) on each image were then digitized using tpsDig (http://life.bio.sunysb.edu/morph/). We chose landmarks that represented reproduciblepoints around the body and the head/opercular region (modified from Tobleret al., 2011). This allowed us to quantify body shape and size, as well asheuristically examine relative head size in relation to body size viadeformation plots.

Shape variation was analyzed using Procrustes PCA, mathematicallyequivalent to performing an unweighted (with respect to bending energy)relative warps analysis (Zelditch et al., 2012). One of the most widelyused programs for geometric morphometric analysis of landmark data,tpsRelw (http://life.bio.sunysb.edu/morph/), uses unweighted relativewarps by default, so these results will be identical. Procrustes PCA iscomputationally simpler and more flexible for further statistical analysisof the resulting morphospace. Landmark data was subjected to aProcrustes superposition, which aligns all specimens, removingdifferences due to size, translation, and rotation. The Procrustes shapecoordinates were then subjected to a covariance-based principalcomponents analysis. This yielded a morphospace composed of aseries of orthogonal, variance-optimized axes, each describing someaspect of shape variation in the sample. Variation along these axes wasmodeled using thin plate splines to visualize the nature of shape variationalong selected axes (in particular, those that showed separation betweenpopulations and/or sexes).

In order to focus on some of the finer scale shape changes, we conductedan additional Procrustes PCA analysis on the male only dataset. Significantdifferences in shape variables were assessed using several methods in the Rstatistical package (R Core Team, 2013). For this analysis, we focusedspecifically on the first seven principal component axes, as they accountedfor approximately 90% of the variance. We assessed the data for interactioneffects, but determined that interaction between PC1-7 were not significant(data not shown). This allowed us to proceedwith a simple linear model with

Fig. 8. Sixteen landmark positions used for alandmark-based analysis. Pictured is a male from thefreshwater population of La Raqueta. We digitized thefollowing landmarks: (1) the tip of the upper jaw, (2) theanterior, (3) center, and (4) posterior most edge of theeye, (5) the posteriodorsal tip of the supraoccipital crest,(6) the anterior and (7) posterior insertion of the dorsalfin, (8) the dorsal and (9) ventral insertion of the caudalfin, (10) the posterior and (11) anterior insertion of theanal fin, (12) the anterior junction of the pelvic fins andthe ventral midline, (13) the ventral most junction ofoperculum and the body midline, (14) the posterior mostedge of the operculum, (15) dorsal and (16) ventralinsertion of the pectoral fin.

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a covariate approach. We assessed the marginal means (means of thegroups adjusted for the covariate of centroid size) for the groups usingGLHT in multcomp (http://multcomp.r-forge.r-project.org/). We alsoassessed differences between groups usingMANOVA as implemented in R.

AcknowledgementsWewould like to give special thanks to colleagues at both the University of La Verne,as well as the University of Colorado for their input and guidance. Thanks to VanessaMorales, Corrina Cavazos, Kristal Castellanos, Kanya Chrisco, Robert Guralnick,Andrew Martin, David Stock, and Dena Smith for assistance with analyses andreading drafts. Thanks also to our Dominican colleagues Carlos Rodriguez, MiguelLandestoy, Marcos Rodriguez, and Patricia Torres for assistance in the field and thelaboratory. Additional thanks to the two anonymous reviewers of this manuscript fortheir insight and suggestions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsAll authors contributed to thewriting and editing of themanuscript. P.F.W., A.C., K.F.W.,and J.V.G. developed the concepts and approach. P.F.W. and A.M. collected andphotographed the fish samples. O.T. and J.V.G. performed the western blot analyses.P.F.W. and J.K. performed the geometric morphometric analysis.

FundingFunding for this project was made possible by a University of La Verne FacultyResearch Grant, a University of Colorado Boulder Museum Research Grant, and aUniversity of Colorado Boulder Department of Ecology and Evolutionary Biologydepartmental graduate student grant.

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